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In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis

[Image: see text] Biopharmaceuticals such as protein and peptide-based drugs are often produced by fermentation processes where it is necessary to monitor the amount and quality of the product expressed during fermentation and for release testing of the final drug product. Standard procedures involv...

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Autores principales: Pedersen, Morten E., Østergaard, Jesper, Jensen, Henrik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2020
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227040/
https://www.ncbi.nlm.nih.gov/pubmed/32426609
http://dx.doi.org/10.1021/acsomega.0c00791
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author Pedersen, Morten E.
Østergaard, Jesper
Jensen, Henrik
author_facet Pedersen, Morten E.
Østergaard, Jesper
Jensen, Henrik
author_sort Pedersen, Morten E.
collection PubMed
description [Image: see text] Biopharmaceuticals such as protein and peptide-based drugs are often produced by fermentation processes where it is necessary to monitor the amount and quality of the product expressed during fermentation and for release testing of the final drug product. Standard procedures involve surface-based ligand binding technologies such as enzyme-linked immunosorbent assay and biolayer interferometry, or extensive purification using, e.g., preparative chromatography followed by spectrophotometric protein quantification. The multistep nature of these methodologies leads to lengthy protocols and renders real-time process control impractical. Recently, flow-induced dispersion analysis (FIDA) was introduced as a novel in-solution ligand binding technology, requiring only nano/microliter sample volumes. FIDA is based on Taylor dispersion analysis in narrow fused silica capillaries and provides the hydrodynamic radius of the binding ligand and complex in addition to the detailed binding characterization. Here, we demonstrate the use of FIDA for quantification of monoclonal IgG antibodies (rituximab) directly in mammalian cell fermentation broth with only 4 min of analysis time. The FIDA assay utilizes a small anti-IgG affibody, conjugated to a fluorophore, as a selective rituximab binder. The apparent change in the hydrodynamic radius of the affibody, as it interacts with known concentrations of rituximab, is used for generating a binding curve in a blank fermentation medium, and hence determining the dissociation constant and complex size. Finally, the binding curve is utilized for quantifying the rituximab titer concentration in clarified fermentation broth samples.
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spelling pubmed-72270402020-05-18 In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis Pedersen, Morten E. Østergaard, Jesper Jensen, Henrik ACS Omega [Image: see text] Biopharmaceuticals such as protein and peptide-based drugs are often produced by fermentation processes where it is necessary to monitor the amount and quality of the product expressed during fermentation and for release testing of the final drug product. Standard procedures involve surface-based ligand binding technologies such as enzyme-linked immunosorbent assay and biolayer interferometry, or extensive purification using, e.g., preparative chromatography followed by spectrophotometric protein quantification. The multistep nature of these methodologies leads to lengthy protocols and renders real-time process control impractical. Recently, flow-induced dispersion analysis (FIDA) was introduced as a novel in-solution ligand binding technology, requiring only nano/microliter sample volumes. FIDA is based on Taylor dispersion analysis in narrow fused silica capillaries and provides the hydrodynamic radius of the binding ligand and complex in addition to the detailed binding characterization. Here, we demonstrate the use of FIDA for quantification of monoclonal IgG antibodies (rituximab) directly in mammalian cell fermentation broth with only 4 min of analysis time. The FIDA assay utilizes a small anti-IgG affibody, conjugated to a fluorophore, as a selective rituximab binder. The apparent change in the hydrodynamic radius of the affibody, as it interacts with known concentrations of rituximab, is used for generating a binding curve in a blank fermentation medium, and hence determining the dissociation constant and complex size. Finally, the binding curve is utilized for quantifying the rituximab titer concentration in clarified fermentation broth samples. American Chemical Society 2020-04-28 /pmc/articles/PMC7227040/ /pubmed/32426609 http://dx.doi.org/10.1021/acsomega.0c00791 Text en Copyright © 2020 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Pedersen, Morten E.
Østergaard, Jesper
Jensen, Henrik
In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title_full In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title_fullStr In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title_full_unstemmed In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title_short In-Solution IgG Titer Determination in Fermentation Broth Using Affibodies and Flow-Induced Dispersion Analysis
title_sort in-solution igg titer determination in fermentation broth using affibodies and flow-induced dispersion analysis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227040/
https://www.ncbi.nlm.nih.gov/pubmed/32426609
http://dx.doi.org/10.1021/acsomega.0c00791
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